The Wg pathway acts in several patterning processes, eliciting varied
responses. In fact, even in the same tissue, Wg input elicits distinct
responses at different times. For example, early in embryogenesis, Wg
input consolidates parasegmental boundaries by maintaining Engrailed
(En) expression in adjacent epidermal cells. Late in embryogenesis, Wg input is no
longer needed for En maintenance, but rather specifies cell fate. In
the ventral epidermis, it specifies the smooth cuticle cell type. It accomplishes this specification by
repressing expression of genes required for denticle fate specification,
including veinlet (ve; also known as
rhomboid), serrate, and shaven-baby. In the dorsal epidermis, Wg specifies a
fine hair cell type but the genes mediating this response are not known. The molecular basis for the stage-specific response to Wg signaling is unclear. Because two redundant receptors Frizzled
and Frizzled2 mediate all Wg signaling,
specificity is not likely to be conferred by multiple receptors with
distinct specificities. Thus, transducers of Wg signaling may trigger
specific responses by interacting with tissue-specific regulatory
proteins, and/or signal transducers activated by another pathway. One such example is the gene teashirt (tsh),
that modulates Wg signaling by binding the transcription regulatory
domain of Armadillo. Lin is essential for late Wingless signaling activity, acting
downstream of Armadillo but upstream of Wg target genes. Because Lin is not required for the early role of Wg signaling, its requirement for late Wg-dependent cell-type specification is stage specific. Moreover, because Lin
is required only minimally for late signaling ventrally, but is essential dorsally, its role in Wg-dependent cell-type specification is tissue specific, or specific to a subregion within a tissue. Thus Lin is used at developmentally different times and
places to modulate the effects of different factors (Hatini, 2000).

The fly embryonic body plan is subdivided into parasegmental units.
Dorsally, rows of epidermal cells adopt four distinguishable cell
fates, 1°-4° (primary to quaternary), depending on their position along the parasegment, generating a precise and reproducible cuticle pattern. A
single row of cells differentiates large, pigmented denticles: the
1° fate. This row is followed by two to three cell rows producing smooth cuticle: the 2° fate. The next two to three rows secrete pigmented thick hairs, the 3° fate, which are shorter than 1° denticles. The following seven to eight rows secrete fine hairs, the
4° fate, which are longer and less pigmented than 3° cells. Three
to four cell rows of smooth cuticle complete the pattern. These cell
types reflect alternative fate decisions made by the underlying
epithelial cells. Wg and Hh inputs specify these fates and organize the
pattern. Hh and
the homeodomain protein, Engrailed, are co-expressed in stripes, the posterior row of
which produces the 1° fate, whereas the anterior rows adopt the smooth
fate. Hh is required across half the parasegment, where cells adopt the
1°-3° fates. When late hh function is blocked, the
1°-3° fates are missing and are replaced by excess 4° fates. Wg is also required across half
the parasegment, where cells adopt the 4° fate. Wg is expressed in the anterior, adjacent to the En/Hh domain, in a subset of
the cells producing the 4° cell type. To
distinguish a possible role for Wg in specifying fate from its earlier
role in maintaining En/Hh expression, two
different conditions were used, each of which provides for the Wg-dependent
maintenance of En/Hh but inactivates or reduces Wg
signaling during fate specification. (1) A
wg temperature-sensitive allele was inactivated after sufficient Wg signaling
had been delivered to maintain En/Hh [6 hr after egg
laying (AEL)]. (2) Dominant-negative Pangolin (Pan, the intracellular transducer of the wingless signal) was expressed using
Ptc-GAL4, which blocks Wg signaling only in cells flanking the
En/Hh domain. When late Wg function is blocked in either
of these ways, the 4° cell fate is missing, and the anterior En cells adopt ectopic 1° fate. These cells are flanked anteriorly by smooth
cuticle. The parasegment is also narrower, suggesting cell loss. In
addition, upon careful examination of these mutant embryos,
occasionally it has been found that the domain of 3° cell fates is extended.
Compared with two to three rows of 3° fate in wild type,
five to six rows are produced in embryos blocked for Wg signaling. To identify additional components mediating Hh and Wg
function, a screen was carried out of an existing mutant collection. The
lin mutation was selected because the 4° fate is missing,
and the domain producing the 3° cell fate is extended from two or
three rows to five or six. In addition, the anterior
En-expressing cells adopt 1° fate instead of smooth cuticle, and smooth cuticle is produced anterior to these
cells. Thus, the pattern in lin mutants is reminiscent of the
pattern in embryos blocked for late Wg signaling, suggesting a role for lin in the Wg pathway (Hatini, 2000).

Because Wg expression decays in lin mutants, it was asked
whether Wg decay could explain the pattern defects. However, Wg
expression decays at 10 hr AEL, while Wg activity is required earlier, between 6 and 9 hr
AEL, for dorsal patterning. Nevertheless, it was
tested directly whether restoring Wg, by driving UAS-Wg using
Arm-GAL4 in lin mutant embryos can restore dorsal patterning,
and it does not. As expected, activating Wg signaling in
wild-type embryos affects the pattern. Because targeted expression of Wg cannot restore the lin phenotype, it is concluded that
lin is required downstream of Wg expression (Hatini, 2000).

The pattern defect in dorsal epidermis suggests that lin
mutations affect only late-stage Wg signaling. Because a maternal contribution of lin could mask an earlier role in Wg
signaling, embryos were examined that lack both maternal and zygotic
lin activity. However, the cuticle phenotype of these embryos
is indistinguishable from zygotic lin mutants.
Thus, lin is necessary only for late, Wg-dependent cell-type
specification in dorsal embryonic epidermis (Hatini, 2000).

Lin and late Wg activities are required for 4° but not
1°-3° cell fates. To determine whether Lin and late Wg function
are sufficient for specifying the 4° cell fate, Arm-GAL4 was used to drive ubiquitous expression of either Lin, Wg, or activated Wg signal transducers. Expression of Wg or activation of the Wg pathway globally
by driving activated Arm expression leads to the replacement of
1°-3° cell fates with 4° fates, consisting of secreted
fine hairs that are longer and less pigmented than 3° cells.
Similarly, driving of global Lin expression elicits an identical
response, replacing the 1°-3° fates with 4° fates.
Thus, Wg and Lin are sufficient for specifying the 4° cell fate when
provided at higher levels. One portion of the parasegment
is less sensitive to ectopic Lin or Wg activity. Driving Lin or Wg
to high levels of expression in cells posterior to the En domain through the use of
Ptc-GAL4 is sometimes not sufficient to transform the 2° fate to
4°. This domain is where Hh activity is normally highest,
suggesting that Hh signaling competes with Lin and Wg in fate selection (Hatini, 2000).

Lin may contribute to Wg signaling either by controlling the
production of a signal or by transducing a signal. These possibilities can be distinguished, because if Lin acts by producing a signal,
restoration of restricted expression of Lin in lin mutants will restore the 4° cell fate non-autonomously. If Lin is required for transducing the signal, the 4° fate will be restored
autonomously only in cells expressing Lin. In lin mutants, the
anterior- and posterior-most En rows differentiate the 1° denticle
type, while two to three cell rows internal to the En domain
differentiate smooth cuticle. Use of
En-GAL4 and UAS-Lin to target Lin to the En domain transforms all
the En-expressing cells cell-autonomously to the 4° cell fate. Cells flanking the En domain are not affected. Similarly,
targeting of Lin to the Wg domain in lin mutants by use of
Wg-GAL4 restores the 4° fate, but only in that portion of
the 4° field that expresses Wg. Two to three rows of cells anterior
to the Wg domain still adopt 3° fate.
Restoration of Lin activity in Wg-producing cells does not restore the
full pattern. Thus, these two experiments indicate that Lin generally
does not regulate expression of a signal, but rather acts
cell-autonomously to specify the 4° cell fate (Hatini, 2000).

There is one instance where Lin may regulate a signal. Expression of
Lin in the Wg domain rescues the ectopic 1° denticle row found in
the En domain of lin mutants. Thus, Lin has a second
role within Wg-expressing cells, and, in this case, it acts
non-autonomously to specify the (smooth) fate of the posteriorly adjacent row of En cells (Hatini, 2000).

Evidence for the suggestion that Lin acts in some way to specify dorsal cell fate
has to this point relied on cuticle analysis, which represents the final
differentiated state of the cells, first visible at about 13 hr AEL.
However, Wg signaling specifies these cell fates between 6 and 9 hr AEL. Thus, to test whether or not Lin acts in
concert with Wg, earlier molecular markers for Wg
patterning need to be identified and the effects of Lin activity on these markers need to be tested. The first Wg-dependent target gene is wg itself. If Wg function is blocked late by expression of dominant-negative Pan with the Ptc-GAL4
driver, late wg expression is lost from both the dorsal and
the ventral epidermis. Reciprocally, if Wg
signaling is activated by driving of activated Arm expression with the
Ptc-GAL4 driver, an ectopic Wg stripe is induced posterior to the En
domain. Thus, wg gene expression depends on Wg input
and provides a molecular readout for the pathway. In lin
mutants, late wg expression fades from the dorsal epidermis of
fully retracted embryos (10 hr AEL). Reciprocally, overexpression of Lin
using the Ptc-GAL4 driver activates wg expression posterior
to the En domain in the dorsal epidermis. The ectopic
expression of wg is identical to that obtained by expression of activated Arm posterior to the En domain. The only distinction is
that the effect of Lin is restricted to the dorsal epidermis, while
global activation of Wg signaling affects the ventral epidermis as well. Thus, Wg input and Lin are both necessary and sufficient for activation of wg gene expression in dorsal epidermis (Hatini, 2000).

The second Wg-dependent target gene is ve (veinless or rhomboid), which is expressed
in a row of cells posteriorly adjacent to the
En/Hh-expressing cells. This spatially restricted pattern is regulated in ventral
epidermis by Wg signaling. Wg regulates ve expression dorsally as well.
For example, if Wg function is inactivated at late stages, ve
is ectopically expressed anterior to the En domain.
Reciprocally, if the Wg pathway is broadly activated, ve
expression is repressed. Thus, ve expression also
provides a molecular readout for the Wg pathway. In lin mutant
embryos, a second stripe of ve is induced anterior to the
En/Hh cells in the dorsal epidermis. Thus, Lin and Wg function are similarly required to repress ve gene expression. It is concluded that Lin acts in concert with Wg in regulating target genes
and consequently patterning the dorsal cell types (Hatini, 2000).

Because Lin acts cell-autonomously upstream of Wg target genes, it was
determined where along the Wg signaling pathway Lin function is
required. This determination was accomplished by forcing activation of
cytoplasmic Wg signal transducers in lin mutants and testing cuticle pattern and ve gene regulation. Wg
signaling was activated by either overexpressing Dishevelled or using
constitutively activated Arm, with the UAS-GAL4 system. Activation of Wg signaling in wild-type embryos
leads to global specification of the 4° cell fate across the dorsal
epidermis. In contrast, this specification does not occur if
lin function is removed. In fact, the
pattern resembles the lin mutant pattern. For example, rather
than adopting 4° fate, 1° and 2° fates are still established,
as is the ectopic 1° fate and the smooth fates anterior to these
cells. Cells in the remainder of the parasegment resemble
immature 3° rather than 4° cells. For example, the cuticle
protrusions are shorter and more pigmented than 4° cells, but their
base is narrower than fully mature 3° cells. The expression of a
molecular marker is consistent with the cuticle pattern, because now
ve is not repressed by increased Wg signaling if lin
activity is removed. Ventrally, Wg activation still
represses ve even when lin activity is removed, further demonstrating the restriction of the role of Lin to dorsal Wg
signaling. Thus, in dorsal epidermis, Lin is crucial for completing Wg
signal transduction, acting downstream or in parallel to Arm (Hatini, 2000).

The epistasis also is consistent with Lin being a downstream
target gene of the Wg pathway. However, this model is unlikely since
lin is still expressed in embryos blocked for Wg signaling, and lin expression is not up-regulated in embryos globally
activated for Wg signaling. Therefore, Lin could act
either in a parallel pathway to the Wg pathway, or may cooperate with Wg signal transducers (Hatini, 2000).

If Lin is acting strictly in parallel to Arm, then both Wg input and
Lin must act for normal patterning. If, however, Lin is required
downstream of Wg signaling then overexpression of Lin in embryos
blocked for Wg signaling will bypass the need for Wg input and restore
Wg-dependent readouts. The dorsal epidermis in embryos null for Wg
signaling is poorly differentiated and occasionally missing. Expression
of Lin in these embryos does not rescue the pattern.
Because lin is not needed for early Wg signaling perhaps it is
not surprising that Lin is not capable of restoring En expression in
the absence of Wg. Expression of Lin in embryos blocked for late Wg
signaling may not allow distinguishing between the two models posed
above because these embryos might contain residual Wg signaling
activity. To examine this issue, Lin was overexpressed using Ptc-GAL4
in wgts embryos shifted to the nonpermissive
temperature at 6 hr AEL, or in embryos co-expressing Lines
and dominant-negative Pan, and their cuticle pattern was examined. In
either experiment, the usual effect on patterning caused by the defect
in Wg signaling is blocked in embryos co-expressing Lin because the 4°
cell fate is restored. For example, anterior to
the En domain, smooth cuticle is replaced with the 4° type. In addition, the 4° cell fate is restored posterior to
the 2° domain. Expression of Lin in embryos expressing
dominant-negative Pan with the ubiquitous Arm-GAL4 driver replaces all
cell fates with 4° cell fate. This result may have two
interpretations. High levels of Lin may trigger a response
independent of Wg input. Alternatively, any residual Wg signaling
that may be present in these embryos allows Lin to function (Hatini, 2000).

In the current model for Wg signaling, Arm regulates Wg target gene
expression in association with the DNA-binding protein Pangolin. Because the
epistasis shows that Lin regulates Wg target gene expression downstream
of Arm, Lin protein would be expected to be found in nuclei. Crude and purified
polyclonal antisera weakly detects Lin protein by Western blot from
embryonic extracts, but these sera fail to detect the
protein in situ. The lin genomic rescue
transgene was modified to include a Myc tag at the carboxyl terminus of the Lin
protein. The modified transgene rescues lin mutants to
adulthood; however, Myc-tagged Lin
protein could not be detected in situ. It is concluded that Lin is expressed below the detection
levels of both the anti-Lin and the commercial anti-Myc antibodies.
However, by use of the UAS-GAL4 system, Lin protein can be detected by antibodies in situ in embryos overexpressing Lin. For instance, embryos carrying UAS-Lin and the
Ptc-GAL4 driver show the expected high levels of Lin protein both
anterior and posterior to the En expression domain at 10hr AEL. However, Lin exhibits a different subcellular localization in each
expression domain. Lin is nuclear in cells anterior to the En domain, in exactly those cells dependent on Lin and Wg function.
Lin is preferentially cytoplasmic posterior to the En domain,
which may help explain why excess Lin sometimes does not alter
patterning in this domain. However, it must be assumed that low
levels of Lin do enter the nucleus, as sometimes these cells are
transformed to the 4° fate. Expression of Lin with Arm-GAL4 shows
that Lin is nuclear in half of the parasegment anterior to the En
domain, and cytoplasmic in the other half posterior to the En domain (Hatini, 2000).

To determine whether the differential localization is signal-dependent
Lin was co-expressed with Wg to ectopically activate Wg signaling. In
this case, Lin accumulates exclusively in nuclei both anterior and posterior to the En domain. To
decrease Wg signaling, dominant-negative Pan was expressed with
Ptc-GAL4. Due to feedback regulation,
this expression leads to reduced Wg expression, and therefore reduced
Wg signaling. In such embryos, the differential localization of Lin is
altered, but mostly at lateral positions in the embryo, where Lin
occasionally localizes to the cytoplasm, anterior to the En domain. In the dorsal epidermis, Lin is still nuclear,
which is consistent with the fact that although Pan function is
compromised in these embryos, excess Lin still promotes the 4° fate.
It is concluded that Wg signaling can influence the subcellular
localization of Lin. In addition, the nuclear localization of Lin
supports the genetic model that Lin is needed to complete transduction
of the Wg signal, probably by cooperating with Arm/Pan in
the nucleus. Interestingly, in the ventral epidermis, where Lin is
required on either side of the En domain, Lin is nuclear both anterior
and posterior to that domain. Thus, Lin subcellular
localization is correlated with its spatial requirement (Hatini, 2000).

Three models are proposed for
Lin function in Wg-dependent cell-fate specification.
(1) Because Lin protein is nuclear in cells in which it acts,
Lin may directly regulate the transcription of Wg target genes by
interacting with DNA or DNA-bound proteins. For instance, Arm and Pan
may act at one site on target genes, while Lin acts directly at an
adjacent site. Because Lin does not have an obvious DNA-binding domain,
it may act in combination with another DNA-binding protein. Pan bound
to one site could easily be imagined to modulate the function of Lin at
a second site, since LEF1/TCF proteins related to Pan can
play an architectural role, regulating the assembly of multiprotein
enhancer complexes. This scenario is reminiscent of
the midgut Ubx enhancer, which contains a Wg-responsive Lef-1
DNA-binding site, and a Dpp-dependent cyclic AMP-responsive element. The identification of response elements in the
target genes ve and wg will be needed to investigate
whether the regulation by Arm/Pan and Lin is direct and
to reveal the precise mechanism at work. It is also possible that Lin
interacts directly with Pan and/or Arm, rather than
participating in a distinct complex. However, initial experiments with
the two-hybrid system or immunoprecipitation have not provided evidence
for direct interactions with Arm (Hatini, 2000).

(2) In a second model, Lin may regulate transcription by creating a state
permissive for Wg signal transducers to act. For example, Lin itself
may act by remodeling chromatin structure, allowing efficient access of
transcription factors to Wg-regulated promoters, leading to an
increased activity of DNA-bound Pan/Arm complexes (Hatini, 2000).

(3) Finally, Lin may regulate transcription factor activity by affecting
the modification state of factors such as Arm and or Pan. Precedent
exists for this: in Caenorhabditis elegans, the Lit1 MAP
kinase regulates the function of Wrm1 and Pop1, proteins related to Arm
and Pan, respectively (Hatini, 2000 and references therein).

Although Lin can promote Wg signaling in the dorsal epidermis, it is
not sufficient to do so ventrally. Thus, Lin can promote Wg signaling
only in a competent region of cells. The activity of Lin dorsally must
depend on additional factors that provide this region with the
competence to modulate Wg signaling in response to Lin. Such factors
could provide a biochemical link between Lin and the Arm/Pan complex (Hatini, 2000).

Whichever model holds, note that the role of Arm and Pan is more
clearly understood for transcriptional activation events, where Arm is proposed to confer an activation function to
the otherwise negative regulator Pan. Only a few genes have been characterized
as being repressed by Wg signal transduction, for example, dpp
and wg in developing leg and wing, respectively, and
veinlet. Unfortunately, response elements
mediating Wg-dependent repression have not yet been identified. It is
likely that the Arm/Pan complex can recruit repressors or
activators to Wg target genes, perhaps in a manner analogous to Smad
proteins, which can activate by recruiting the co-activator
CBP/p300 or repress by recruiting co-repressors such as TGIF or Ski, which, in turn, bind histone deacetylases. The identification of ve as a gene repressed by Wg input will eventually provide tools to investigate negative regulation by the Wg pathway (Hatini, 2000).

Because Lin is essential for Wg-dependent cell-type specification
acting downstream or in parallel to Arm, and because Lin nuclear
localization is influenced by Wg, a particularly attractive model
involves Arm in the localization of Lin.
Arm/beta-catenin share homology with the
importin/karyopherin family of transport receptors and
can bind the nuclear pore machinery (Fagotto, 1998), raising the
possibility that one function of Arm/beta-catenin is to
import regulatory proteins to the nucleus. Perhaps at the same time as Wg signaling stabilizes Arm, Wg associates with Lin in the cytoplasm and transports Lin across the nuclear membrane. The association of Arm with other regulatory proteins may also promote their nuclear import. Alternatively, because the
phosphorylation state of a protein can lead to transport into or out of
the nucleus, perhaps Wg signaling affects Lin localization through the
inhibition of the serine/threonine kinase Shaggy. In this
case, Lin nuclear import would be regulated by an upstream component of
the Wg pathway and then interact with other downstream components
(Arm/Pan) in the nucleus (Hatini, 2000).

Wg signaling has two distinct roles in patterning the embryonic
epidermis. The first is to prevent an En-dependent signal from crossing
over anteriorly. When Wg signaling is deficient, a signal from the
En domain, such as Hh, crosses over and specifies cell fate anterior to
it, creating a mirror image ve expression and cuticle pattern
(smooth-1°/1°-smooth). In lin mutants,
ve expression and pattern are similarly reorganized,
indicating that lin is necessary for this role of Wg. The
second role of Wg is to specify cell fate in half of the parasegment,
an event that is also blocked in lin mutants (Bokor, 1996). Thus, these two patterning roles of Wg are compromised
in lin mutants (Hatini, 2000).

Normally, Hh patterns posteriorly from its source, specifying the
1°-3° cell fates, whereas Wg patterns to the anterior,
specifying the 4° cell fate. The domains of Wg and Hh influence meet
in the middle of the parasegment, at approximately the interface of
3° and 4° cell types. A balance between Wg and Hh signaling is
essential for proper patterning in this region since changing the strength of either can alter the pattern in the middle of the parasegment. For
example, reduction of Wg function results in excess 3° cells at the
expense of 4° cells. Similarly, a gradual increase in Hh
signaling leads to a gradual increase in the number of 3° cells at
the expense of 4° cells. It is proposed that Lin helps govern the balance between Wg and Hh
influence by promoting Wg signaling activity, which can thereby better
compete with Hh function. An increase of Lin activity can alter this
balance, leading to excess 4° cells at the expense of 3° cells. At higher levels of Lin, the entire domain under Hh
influence differentiates as 4° cells. Aside from the
contribution of Lin levels to the pattern, the level of Hh is also
crucial. Hh may contribute to this balance by antagonizing Lin
function. The
antagonism will be highest posterior to the En domain where Hh
signaling is the highest. In fact, further evidence is found for
this antagonism, because the expression of high levels of Lin in this region is
not always sufficient to replace the 2° with 4° fate (Hatini, 2000).

It is proposed that, in wild type, Hh antagonizes Lin activity in the
middle of the parasegment, thereby toning down Wg signaling activity in
this region, and allowing these cells to adopt 3° rather than 4°
fate. One way by which Hh may antagonize Lin function, and thereby Wg
signaling, is by exporting Lin to the cytoplasm (Hatini, 2000).

GENE STRUCTURE

mRNA length - 3416

Bases in 5' UTR - 280

Bases in 3' UTR - 559

PROTEIN STRUCTURE

Amino Acids - 858

Structural Domains

Lines is a novel protein with no similarity to any other characterized proteins in the NCBI database (Hatini, 2000).